Ocular irritation testing is extremely relevant to assuring adequate safety levels of public health as new formulations of chemicals and products are introduced. In most cases, these safety assessments are performed using the Draize Rabbit Eye test, resulting in thousands of rabbits used in testing every year. Alternatives have been discussed since the 80s without any appreciable acceptance from regulators. As more governmental agencies mandate the replacement of using live animals in regulatory safety assessment of consumer products, chemicals and cosmetics, the need for a complete alternative solution to ocular irritation testing grows. Currently, there is no single or set of tets that have been validated and accepted by regulatory agencies as a full replacement for rabbit ocular irritation testing. The aim of this project is to evaluate the applicability of the Integratd In vitro/Alternative Ocular (IIVAO) pronounced -/eye vay o/ Toxicology Testing Strategy to categorize potential ocular irritants into regulatory toxicity classes using four integrated in vito and alternative ocular irritation assays. IIVAO will demonstrate it is possible to effectively replace mandated acute ocular irritation testing using the Draize Rabbit Eye Test with non-animal test methods. For the purpose of this project, we have focused on assessing the performance of IIVAO using Environmental Protection Agency (EPA) testing classifications. IIVAO, an integrated testing strategy (ITS), comprises in vitro and alternative tests that have been chosen for their ability to effectively detect specific ranges of ocular irritation, which correspond to regulatory classifications of ocular irritation/corrosivity - the Chorioallantoic Membrane Vascular Assay (CAMVA), the Bovine Cornea Opacity/Permeability test (BCOP), the Porcine Cornea Opacity/Reversibility Assay (PorCORA), the MatTek EpiOcular(EO)-3D human tissue construct and the Porcine Confocal Assay (PorFocal). This grant project will confirm and validate a comprehensive testing protocol that allows for a thorough evaluation and categorization of test materials for ocular irritancy potential ranging from non-irritating to oculr corrosion without using live animals. We intend to perform validations of each assay and collectively as an integrated system using a test set of chemicals that are well characterized for specific categories of ocular irritancy. Additionally, we propose to utilize the finalized integratd testing protocol to evaluate a number of `Off-The-Shelf' commercial products for those categories of ocular irritancy to demonstrate the effectiveness of the IIVAO testing strategy with mixtures and final product formulations. The long-term project goal is to submit the IIVAO testing strategy to the EPA for consideration as a standalone alternative to the Draize Rabbit Eye test. Validation and acceptance of IIVAO will significantly reduce the number of rabbits used in the toxicological assessment of consumer products, chemicals and raw materials by replacing rabbits with an ITS of in vitro and alternative assays.